Neuropathic pain refers to alterations in pain perception (spontaneous pain, hyperalgesia and allodynia) that arise from lesions in the nervous system. It affects a large sector of the population. Veterans are especially susceptible to neuropathic pain, because of their exposure to combat wounds and diabetes. Neuropathic pain is notoriously difficult to treat. It responds poorly to opiates, probably because of a loss of their analgesic effect in the spinal cord. A key site of pain modulation is the synapses between nociceptive afferents and dorsal horn neurons. In particular, substance P-containing afferents that make direct synapses with dorsal horn neurons that project to the brain. The receptor for substance P, the neurokinin 1 receptor, is present in these neurons and produces an increase in their excitability responsible for hyperalgesia. Moreover, since there is a single synapse between the substance P-containing afferents and the brain, changes in transmitter release in this synapse have important effects on pain. This project will measure substance P release as an indicator of overall activity of these synapses, investigating how it is inhibited by two receptors present in primary afferent terminals: the 5-opioid receptor and the 12 adrenergic receptor. Recent studies in cell cultures and our data indicate that 5-opioid receptors inhibit neurotransmitter release by a mechanism unique of primary afferents: phosphorylation of voltage-gated calcium channels by Src family kinases. In contrast, 12 receptors inhibit these calcium channels by the mechanism commonly found in other neurons: binding of G protein 23 subunits (G23). Preliminary data also show that substance P release is inhibited by 5-opioid receptors in normal rats but not in neuropathic rats, which parallels the lack of efficacy of opioids in neuropathic pain. In contrast, 12 receptor agonists do not lose their efficacy in neuropathic pain. We formulated the following hypotheses: 1) 5-opioid and 12 adrenergic receptors inhibit SP release by inactivating Ca(V) channels; the effect of 12 receptors is mediated by G23 while that of 5-opioid receptors is additionally mediated by SFKs; 2) in neuropathic pain, inhibition of SP release by 5-opioid receptors is loss, but its inhibition by 12 adrenergic receptors and synergy between 5- opioid and 12 receptors are still present. The Specific Aims of this project are: 1) to study the mechanisms by which 5-opioid and 12 receptors inhibit substance P release; 2) to study changes in opioid and adrenergic inhibition of substance P release in neuropathic pain. The project is significant in that it will study unique signaling mechanisms in nociceptive afferents that can become prime targets for developing analgesic drugs. Moreover, it will investigate how these signaling mechanisms undergo specific changes in neuropathic pain. The methodological approach in Aim 1 will consist in using neurokinin 1 receptor internalization as an in situ measure of substance P release, and pharmacological and molecular (small interfering RNA) approaches to dissect the signaling pathways used by the 5-opioid and 12 receptors. In Aim 2, we will use a rat model of neuropathic pain to investigate alterations in these receptors and their signaling systems.